Nucleoside H-phosphonates. 18. Synthesis of unprotected nucleoside 5'- H-phosphonates and nucleoside 5'-H-phosphonothioates and their conversion into the 5'-phosphorothioate and 5'-phosphorodithioate monoesters

Article abstract of DOI:10.1021/jo980491u

A simple and efficient protocol for the preparation of unprotected nucleoside 5'-H-phosphonates and nucleoside 5'-H-phosphonothioates via a one- step deprotection of suitable precursors with methylamine has been developed. The synthetic utility of the unprotected nucleotide derivatives was demonstrated by converting them under mild conditions to the corresponding nucleoside 5'-phosphorothioate and nucleoside 5'-phosphorodithioate monoesters. Factors affecting oxidation of H-phosphonate, H-phosphonothioate, and phosphite derivatives with elemental sulfur are also discussed.

Full text of DOI:10.1021/jo980491u

8150
J . Org. Chem. 1998, 63, 8150-8156
Nu cleosid e H-P h osp h on a tes. 18. Syn th esis of Un p r otected
Nu cleosid e 5′-H-P h osp h on a tes a n d Nu cleosid e
5′-H-P h osp h on oth ioa tes a n d Th eir Con ver sion in to th e
5′-P h osp h or oth ioa te a n d 5′-P h osp h or od ith ioa te Mon oester s
J adwiga J ankowska, Anna Sobkowska, J acek Cies´lak, Michał Sobkowski, and
Adam Kraszewski*
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/ 14,
61-704 Poznan´, Poland
J acek Stawin´ski*
Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University,
S-106 91 Stockholm, Sweden
David Shugar
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawin´skiego 5A,
02-106 Warsaw, Poland
Received March 16, 1998
A simple and efficient protocol for the preparation of unprotected nucleoside 5′-H-phosphonates
and nucleoside 5′-H-phosphonothioates via a one-step deprotection of suitable precursors with
methylamine has been developed. The synthetic utility of the unprotected nucleotide derivatives
was demonstrated by converting them under mild conditions to the corresponding nucleoside 5′-
phosphorothioate and nucleoside 5′-phosphorodithioate monoesters. Factors affecting oxidation of
H-phosphonate, H-phosphonothioate, and phosphite derivatives with elemental sulfur are also
discussed.
In tr od u ction
ses highlighted some fundamental problems connected
with their preparation.
In the last two decades phosphorothioate analogues of
naturally occurring substances have become a firmly
established tool in biochemical and biological studies
directed toward unraveling of enzyme functions and
mechanisms.1 Due to their chirality at the phosphorus
center and higher stability toward enzymatic hydrolysis,
phosphorothioate derivatives have proven to be valuable
as model constructs for designing enzyme inhibitors and
transition state analogues, as stereochemical probes in
elucidation of mechanisms of enzyme-catalyzed phospho-
ryl transfer reactions, or for probing structures of ri-
bozymes, etc.1-3
Although nucleoside 5′-phosphate monoesters are cen-
tral compounds in numerous biochemical and pharma-
cological studies,2,4,5 their phosphorodithio analogues, in
contradistinction to those of phosphate diesters, have
received relatively little attention.6-8 In fact, nucleoside
phosphorodithioates, bearing two sulfur atoms at the
nonbridging positions of the phosphomonoester moiety,
have been prepared only recently6,7,9,10 and their synthe-
Due to the inherent instability of phosphorodithioate
monoesters, most synthetic organic methods for thio-
phosphorylation of nucleosides1 have been found inap-
plicable to the synthesis of these phosphate analogues.
For example, an attempted synthesis of nucleoside phos-
phorodithioates via S-alkyl and O-alkyl phosphorodithio-
ate diesters or the corresponding phosphotriesters failed,
due to problems encountered during the removal of
phosphate protecting groups.6 An approach involving
nucleoside H-phosphonodithioates as intermediates has
been more successful, but also this synthesis was ham-
pered by a low yield in some crucial steps and formation
of significant amounts of side products during the final
deprotection.6 The route via 2-thio-1,3,2-dithiaphos-
pholane derivatives, as proposed by Okruszek et al.,7
produced nucleoside phosphorodithioates in acceptable
yields (ca. 50%), but it required the inclusion of two extra
steps (the introduction and removal of a 2-cyanoethyl
group) into the synthetic protocol to prevent a severe
decomposition of the products during the hydrolytic
opening of the dithiaphospholane ring. The development
of 9-fluorenemethyl H-phosphonothioate as a -(H)P(S)-
(1) Eckstein, F. Annu. Rev. Biochem. 1985, 54, 367-402.
(2) Eckstein, F.; Gish, G. TIBS 1989, 97-100.
(3) Herschlag, D.; Piccirilli, J . A.; Cech, T. R. Biochemistry 1991,
30, 4844-4854.
(4) Mitchell, A. G.; Thomson, W.; Nicholls, D.; Irwin, W. J .; Freeman,
S. J . Chem. Soc., Perkin Trans. 1 1992, 2345-2353.
(5) Perigaud, C.; Gosselin, G.; Lefebvre, I.; Girardet, J .-L.; Benzaria,
S.; Barber, I.; Imbach, J .-L. Bioorg. Med. Chem. Lett. 1993, 3, 2521-
2526.
(6) Seeberger, P. H.; Yau, E.; Caruthers, M. H. J . Am. Chem. Soc.
1995, 117, 1472-1478.
(7) Okruszek, A.; Olesiak, M.; Krajewska, D.; Stec, W. J . J . Org.
Chem. 1997, 62, 2269-2272.
(8) Golos, B.; Dzik, J . M.; Rode, W.; J ankowska, J .; Kraszewski, A.;
Stawin´ski, J .; Shugar, D. Interaction of thymidylate synthase with the
5′-thiophosphates and 5′-H-phosphonates of 2′-deoxyuridine, thymidine
and 5-fluoro-2′-deoxyuridine; In Chemistry and Biology of Pteridines
and Folates; Pfleiderer, W., Rokos, H., Eds.; Blackwell Science: Berlin,
1997; pp 423-426.
(9) Seeberger, P. H.; J orgensen, P. N.; Bankaitisdavis, D. M.; Beaton,
G.; Caruthers, M. H. J . Am. Chem. Soc. 1996, 118, 9562-9566.
(10) J ankowska, J .; Cieslak, J .; Kraszewski, A.; Stawin´ski, J .
Tetrahedron Lett. 1997, 38, 2007-2010.
10.1021/jo980491u CCC: $15.00 © 1998 American Chemical Society
Published on Web 10/28/1998

Nucleoside H-Phosphonates
J . Org. Chem., Vol. 63, No. 23, 1998 8151
O- group transferring reagent9,10 significantly improved
the synthesis of nucleoside phosphorodithioates, although
some degradation of the product during the removal of
the 9-fluorenemethyl group was still observed.10
Nucleoside phosphorodithioates are interesting as
potential inhibitors of several classes of enzymes, e.g.
kinases, phosphatases, polymerases, etc. Preliminary
biological studies indicate that phosphorodithioate mo-
noesters derived from naturally occurring deoxyribo-
nucleosides are resistant toward alkaline phosphatase
treatment,9,11 are neutral against DNA polymerase I
Klenow fragment and human immunodeficiency virus
reverse transcriptase (HIV RT),7,9,11 and are competitive
inhibitors of avian myeloblastosis virus reverse tran-
scriptase (AMV RT)11 and thymidylate synthase.8 Since
unprotected nucleotide analogues are required for most
biochemical applications, an additional factor has to be
taken into account when designing the synthesis of
nucleoside phosphorodithioates, namely, the stability of
the phosphorodithioate function during the final removal
of sugar and heterocyclic base protecting groups.
To find a possibly general solution to the problem of
instability of some functionalities during a final depro-
tection, we have recently embarked on studies directed
toward preparation and evaluation of synthetic potential
of unprotected nucleoside H-phosphonate and H-phospho-
nothioates. Since the H-phosphonate or H-phosphono-
thioate function can be selectively activated under oxi-
dative conditions, access to such precursors would alleviate
most problems often encountered during the synthesis
of some labile nucleotide analogues, e.g. nucleoside
phosphorodithioate monoesters. This kind of approach
would also considerably expand applications of H-phos-
phonate and H-phosphonothioates as synthetic interme-
diates.
phorodithioate monoesters under exceedingly mild reac-
tion conditions.
Unprotected nucleoside H-phosphonate and nucleoside
H-phosphonothioate monoesters seem to be well suited
as synthetic precursors, for at least two reasons. First,
we expected, that these compounds should be equally
stable and easy to handle as their protected counterparts,
and with a variety of phosphonylation14-17 and thiophos-
phonylation10,18,19 methods available, they can also be
relatively easily accessible. Second, being tetracoordi-
nated P(III) compounds, they should undergo a variety
of selective oxidative transformations at the phosphorus
center20 providing, under mild conditions, unprotected
P(V) analogues.
Syn th esis of Un p r otected Nu cleosid e 5′-H-P h os-
p h on a tes a n d Nu cleosid e 5′-H-P h osp h on oth ioa tes.
First, we have been searching for a simple method for
the preparation of nucleoside 5′-H-phosphonates 3a -f
and nucleoside 5′-H-phosphonothioates 4a -f. Due to a
plethora of synthetic methods available for the phosphonyl-
ation14-16 and thiophosphonylation10,18,19 of protected
nucleosides, we decided to use as starting materials for
3 and 4 standard nucleosidic precursors that are either
easy to prepare or are commercially available. To this
end, suitably protected N,O-acylated deoxyribonucleo-
sides were converted into the corresponding 5′-H-phos-
phonates 1a -f using the diphenyl H-phosphonate
method.15 These products without intermediate purifica-
tion were subjected to various deprotection procedures.
Among the protocols investigated, the most convenient
was found to be that of utilizing aqueous methylamine
(50%)21 as a deprotecting reagent. In accordance with
the reported data,21 methylamine proved to be superior
to aqueous concentrated ammonia, both in efficiency and
in time required for a complete deprotection. The most
significant differences between aqueous ammonia and
methylamine as deprotecting reagents were observed for
the deoxyguanosine derivative 1c [1 h, RT (room tem-
perature), for methylamine vs 24 h, 50 °C, for the
concentrated ammonium hydroxide]. Thus, we adopted
treatment with methylamine (1 h, RT) as a standard
deprotection procedure22 for the preparation of unpro-
tected nucleoside 5′-H-phosphonates 3a -f from the cor-
responding precursors 1a -f. Since the deprotection
reactions were virtually quantitative, 3a -f could be
isolated by simple flash silica gel chromatography, which
provided the products of purity >98%, in satisfactory
yields. Structures of the isolated compounds were con-
firmed by spectral and chromatographic analysis [1H and
31P NMR (Table 1) and HRMS and TLC].
In this paper we describe synthesis of various unpro-
tected nucleoside H-phosphonates and nucleoside H-
phosphonothioates and demonstrate that they can serve
as convenient precursors for the preparation of nucleoside
phosphoromonothioate and phosphorodithioate mono-
esters.12
Resu lts a n d Discu ssion
To avoid a possible degradation of a fragile modification
in some nucleotide analogues during a multistep syn-
thesis, we searched for an approach enabling the intro-
duction of the most labile functionality in the last
synthetic step. In the instance of nucleoside phospho-
rothioate monoesters, this would call for unprotected
nucleotidic precursors,13 which could be selectively con-
verted in the desired product. As a viable approach for
this purpose we considered sulfurization of unprotected
nucleoside H-phosphonates and nucleoside H-phospho-
nothioates, which should provide a facile access to
nucleoside phosphoromonothioate and nucleoside phos-
(14) Stawin´ski, J .; Thelin, M. Nucleosides Nucleotides 1990, 9, 129-
135.
(15) J ankowska, J .; Sobkowski, M.; Stawin´ski, J .; Kraszewski, A.
Tetrahedron Lett. 1994, 35, 3355-3358.
(16) Garegg, P. J .; Regberg, T.; Stawin´ski, J .; Stro¨mberg, R. Chem.
Scr. 1986, 26, 59-62.
(17) Marugg, J . E.; Tromp, M.; Kuyl-Yeheskiely, E.; van der Marel,
G. A.; van Boom, J . H. Tetrahedron Lett. 1986, 27, 2661-2664.
(18) Stawin´ski, J .; Thelin, M.; Westman, E.; Zain, R. J . Org. Chem.
1990, 55, 3503-3506.
(11) Seeberger, P. H.; Caruthers, M. H. Tetrahedron Lett. 1995, 36,
695-698.
(12) Studies on synthesis of phosphate analogues utilizing unpro-
tected H-phosphonates, H-phosphonothioates, and H-phosphonodithio-
ates derived from various natural products are currently under
investigation in these laboratories.
(13) Attempted dithiophosphorylation of unprotected (or partially
protected) nucleosides with unsubstituted dithiophosphate dianion in
dimethylformamide afforded exclusively mixtures of the corresponding
3′(5′) nucleoside phosphoromonothioates. See: Dunaway-Mariano, D.
Tetrahedron 1976, 32, 2991-2996.
(19) Zain, R.; Stro¨mberg, R.; Stawin´ski, J . J . Org. Chem. 1995, 60,
8241-8244.
(20) Stawin´ski, J . Some Aspects of H-Phosphonate Chemistry; In
Handbook of Organophosphorus Chemistry; Engel, R., Ed.; Marcel
Dekker: New York, 1992; pp 377-434.
(21) Reddy, M. P.; Farooqui, F.; Hanna, N. B. Tetrahedron Lett.
1995, 36, 8929-8932.
(22) Unprotected nucleoside H-phosphonates 3a -f were stable
under the reaction conditions even during a prolonged (12 h) treatment
with aqueous methylamine (31P NMR and TLC analysis).

8152 J . Org. Chem., Vol. 63, No. 23, 1998
J ankowska et al.
Ta ble 1. 31P NMR Da ta for Nu cleosid e H-P h osp h on a tes,
H-P h osp h on oth ioa tes, Nu cleosid e P h osp h or oth ioa tes,
a n d P h osp h or od ith ioa tes
Ch a r t 1
compd
δP (ppm)a
1J HP (Hz)
3J HP (Hz)
3a
3b
3c
3d
3e
3f
4a
4b
4c
4d
4e
4f
5a
5b
5c
5d
5e
5f
6.54
5.13
6.63
5.00
6.42
6.48
55.40, 55.56c
55.35, 55.54c
55.40, 55.52c
55.23, 55.38c
55.44, 55.64c
55.67, 55.68c
45.09
48.72
44.82
47.72
44.38
639.6
621.0
639.6
619.2
638.7
639.0
598.1, 598.1
593.2, 592.3
594.2, 595.1
592.3, 592.3
594.2, 593.2
575.6, 575.6
6.5b
7.0b
6.0b
6.3b
8.3, 8.3b
d
8.3, 8.3b
8.3, 7.4b
8.5, 7.5b
tion with elemental sulfur,26 we investigated this ap-
proach in more detail and developed it as a general
procedure for the preparation of various unprotected
nucleoside phosphoromono- and phosphorodithioate mo-
noesters.
7.4, 7.4b
e
e
e
e
e
e
Preliminary experiments showed that rates of sulfur-
ization of the silylated nucleoside H-phosphonates varied
significantly with the solvent and the base used for the
reaction. We have previously observed the same phe-
nomena during sulfurization of H-phosphonate diesters.27
These findings suggest that the mechanism involved is
similar to that previously found for the sulfurization of
tertiary phosphines and phosphite triesters,28-30 for
which second-order kinetics and the opening an octa-
atomic sulfur ring in the rate-determining step were
observed. Since these reactions proceed via a polar
transitions state with phosphorus acquiring positive
charge, they are sensitive to the polarity of solvents.
43.47
6a
6b
6c
6d
6e
6f
87.94
87.74
87.95
87.68
88.10
87.92
6.5f
4.6f
7.4f
7.5f
6.5f
6.5f
a
Spectra recorded in H2O containing NaOD (pH ) 8.0) with
b
heteronuclear decoupling (H3PO4 as external reference). Two
triplets. c Two diastereoisomers. Poorly resolved triplet. e Unre-
d
solved broad multiplet. f Triplet.
For the preparation of unprotected nucleoside 5′-H-
phosphonothioates 4, easily accessible nucleoside 9-fluo-
renemethyl H-phosphonothioates10 of type 2 were used.
An attempted deprotection of these compounds with
aqueous methylamine produced, however, a mixture of
products, most likely due to the competing loss of
nucleoside moieties (ca. 25%, 31P NMR). We solved this
problem by carrying out the deprotection of nucleoside
H-phosphonothioates 2 as a two-step “one-pot” reaction.
First, the 9-fluorenemethyl group from 2a -f was re-
moved with anhydrous triethylamine (20 min),10 and the
resulting nucleoside H-phosphonothioate monoesters with-
out their isolation were treated with aqueous 50% me-
thylamine (RT, 1 h)23 to remove the sugar and nucleobase
protecting groups. The unprotected nucleoside 5′-H-
phosphonothioates 4a -f were isolated by silica gel
column chromatography with excellent yields, and their
structures were confirmed by 1H and 31P NMR (Table 1),
HRMS, and TLC analysis.
Syn th esis of Un p r otected Nu cleosid e 5′-P h osp h o-
r om on oth ioates an d Nu cleoside 5′-P h osph or odith io-
a tes. In contradistinction to phosphite triesters and
H-phosphonate diesters, the phosphorus center in H-
phosphonate monoesters is rather resistant to oxida-
tion.20 However, conversion of these compounds into
tervalent silyl derivatives significantly enhanced their
susceptibility toward electrophiles and facilitated their
oxidative transformations.24 Inspired by the Hata et al.25
findings that thymidine bis(trimethylsilyl) phosphite can
produce the corresponding phosphorothioate in the reac-
Due to significant differences in reactivity of tervalent
vs tetracoordinated phosphorous acid esters toward
sulfur, first we wanted to assess the extent of their
formation during silylation of H-phosphonate monoesters
under various reaction conditions. Ethyl H-phosphonate
1
3
7 (δP 4.76 ppm, J HP ) 609.3 Hz, J HP ) 8.5 Hz) (Chart
1), treated with (TMS)Cl (3 molar equiv) in pyridine
produced only the tetracoordinated species, ethyl trim-
1
ethylsilyl H-phosphonate (8) (δP -3.51 ppm, t, J HP
)
689.7 Hz, 3J HP ) 8.6 Hz), which then reacted with added
sulfur (3 molar equiv) with a rate comparable to that of
diethyl H-phosphonate (a few hours) to produce (after
hydrolysis) ethyl phosphorothioate (10) (δP 43.68 ppm,
m, J HP ) 8.5 Hz).31 When the silylation of H-phospho-
3
nate 7 was carried out in pyridine in the presence of
triethylamine (20 mol equiv), the putative tervalent
species, ethyl bis(trimethylsilyl) phosphite (9) (δP 117.86,
3
t, J HP ) 7.3 Hz), was produced as the sole phosphorus-
(26) The reaction was carried out on a small scale and the product
isolated by paper chromatography.
(27) For example, dithymidine (3′-5′) H-phosphonate (0.25 mmol/2
mL) in the presence of triethylamine (2 equiv) underwent oxidation
with sulfur (2 equiv) to the corresponding dinucleoside phosphorothio-
ate within 1 min in DMF or MeOH, ca. 10 min in pyridine, ca. 40 min
in dioxane or toluene, and ca. 90 min in carbon disulfide. When
triethylamine was replaced by diisopropylamine or DBU, the reaction
in dioxane went to completion within 10 and 1 min, respectively (R.
Wallin and J . Stawin´ski, unpublished results).
(28) Bartlett, P. D.; Meguerian, G. J . Am. Chem. Soc. 1956, 78,
3710-3715.
(29) Bartlett, P. D.; Cox, E. F.; Davis, R. E. J . Am. Chem. Soc. 1961,
83, 103-109.
(23) Similarly to unprotected nucleoside H-phosphonates, H-phospho-
nothioates 4a -f were stable under the reaction conditions even during
a prolonged (12 h) treatment with aqueous methylamine (31P NMR
and TLC analysis).
(24) Orlov, N. F.; Sudakova, E. V. Chem. Abst. 1969, 70, 87881e.
(25) Hata, T.; Sekine, M. Tetrahedron Lett. 1974, 3943.
(30) Lloyd, J . R.; Lowther, N.; Zsabo, G.; Hall, C. D. J . Chem. Soc.,
Perkin Trans. 2 1985, 1813-1817.
(31) The reaction proceeded slightly faster when 10 molar equiv of
(TMS)Cl was used, even though no detectable changes in the composi-
tion of the reaction mixture could be observed by 31P NMR spectros-
copy.

Nucleoside H-Phosphonates
J . Org. Chem., Vol. 63, No. 23, 1998 8153
Sch em e 1
containing product. This was much more reactive than
the monosilyl ester 8 and underwent quantitative sul-
furization with added sulfur (3 molar equiv) within 3
min.32
Finally we have run the 31P NMR experiment in which
nucleoside H-phosphonate 3d (0.1 mol/L) suspended in
pyridine was treated with trimethylsilyl chloride ((TMS)-
Cl, 5 molar equiv) in the presence of triethylamine (20
molar equiv). This afforded rapidly (<3 min) the corre-
sponding nucleoside bis(trimethylsilyl) phosphite,33 which
upon addition of sulfur (6 molar equiv) was quantitatively
converted into nucleoside 5′-phosphorothioate 4d (for 31P
NMR chemical shifts and splitting patterns, see Table
1).
As to a possible role of a base during sulfurization of
phosphorus esters, some further observations are perti-
nent. Triphenyl phosphite (11), apparently due to the
electron-withdrawing effect of three phenyl groups, is
rather resistant to oxidation and does not react with
sulfur in toluene30 or in pyridine within several hours.
An analogous reaction when carried out in pyridine in
the presence of triethylamine was much faster and went
to completion within 30 min, producing triphenyl phos-
phorothioate (12) (δP 53.24 ppm). A similar catalytic
effect of triethylamine on sulfurization of tertiary phos-
phines has been previously observed by Bartlett et al.,29
who assigned it to the conversion of octaatomic sulfur
into more reactive polysulfides, mediated by sulfide
anion, generated from hydrogen sulfide present in el-
emental sulfur as impurities. Since we observed that the
catalytic effect was more profound in pyridine than in
toluene, it is apparent that polar solvents favored this
process.34 Thus, a base during sulfurization of H-phos-
phonate derivatives, most likely plays a dual role: (i) it
facilitates abstraction of the P-bound hydrogen to gener-
ate the corresponding tervalent species which are more
prone to oxidation and (ii) activates sulfur via its conver-
sion to more reactive polysulfides.
On the basis of the above results, we elaborated a
general “one-pot” procedure for the synthesis of nucleo-
side phosphorothioates of type 5 from unprotected nu-
cleoside H-phosphonates 3 (Scheme 1). In this approach
substrates of type 3 (1 molar equiv) and sulfur (6 molar
equiv) suspended in pyridine containing triethylamine
(20 molar equiv) were treated with (TMS)Cl (5 molar
equiv). After 5 min, the starting material was completely
converted (as determined by TLC analysis) to the ex-
pected nucleoside phosphorothioates 5, which after simple
workup were isolated in satisfactory yields by means of
silica gel flash chromatography. The structure and
homogeneity of the obtained final products 5a -f were
ascertained on the basis of spectral and chromatographic
analysis [1H and 31P NMR (Table 1), HRMS, and TLC].
cannot be converted into the corresponding tervalent bis-
(silyl) phosphites35 in pyridine, irrespective of excess of
(TMS)Cl and triethylamine used, the produced under
such conditions tetracoordinated monosilyl derivatives36
were reactive enough to undergo smooth sulfurization.
This is in line with the observed higher susceptibility of
H-phosphonothioate diesters to oxidation,37,38 which can
be related to higher acidity of the P-H bond in H-
phosphonothioates vs H-phosphonates.39 We cannot,
however, exclude the intermediacy of tervalent silyl
species (nucleoside O,S-disilyl phosphorothioites), which
may be formed in small concentrations, below the detec-
tion level of 31P NMR spectroscopy. The produced in such
a way nucleoside phosphorodithioates 6a -f were isolated
essentially as described for H-phosphonates of type 3.
One should, however, exercise some caution during the
workup procedure, since these compounds became rather
unstable at pH below 7.5.6
In conclusion, we have developed a simple and efficient
method for the synthesis of nucleoside phosphoromono-
thioates 5 and nucleoside phosphorodithioates 6, utilizing
sulfurization of unprotected nucleoside H-phosphonate
and nucleoside H-phosphonothioates. For the prepara-
tion of the latter ones, we designed a simple synthetic
The same procedure also worked efficiently in the
instance of sulfurization of unprotected nucleoside H-
phosphonothioates 4 to produce quantitatively the cor-
responding phosphorodithioates 6 in less than 5 min.
Although the starting H-phosphonothioate monoesters 4
(35) Bollmark, M.; Stawin´ski, J . Tetrahedron Lett. 1996, 37, 5739-
5742.
(36) Incremental addition of (TMS)Cl to 4d in pyridine (δP ) 53.7
and 53.1 ppm, 1J HP ) 568 and 571 Hz) causes gradual broadening and
shifting the signals toward lower field. With 3 equiv of (TMS)Cl the
1
resonances sharpened again, and their chemical shifts and J HP
(32) Triethyl phosphite in methylene chloride or in pyridine under-
goes sulfurization to produce triethyl phosphorothioate also in less than
3 min (31P NMR experiment).
coupling constants remained unaffected (δP ) 57.0 and 56.4 ppm, 1J HP
) 659 and 660 Hz) upon further addition of the silylating agent or
triethylamine.
(33) Garegg, P. J .; Regberg, T.; Stawin´ski, J .; Stro¨mberg, R. J . Chem.
Soc., Perkin Trans. 1 1987, 1269-1273.
(34) Upon addition of triethylamine to a solution of sulfur in
pyridine, a deep yellow color, characteristic for polysulfides, developed.
This disappeared during the course of the reaction with phosphite 11.
(37) Stawin´ski, J .; Thelin, M.; Zain, R. Tetrahedron Lett. 1989, 30,
2157-2160.
(38) Stawin´ski, J .; Stro¨mberg, R.; Szabo´, T.; Thelin, M.; Westman,
E.; Zain, R. Nucleic Acids Sym. Ser. 1991, 24, 95-98.
(39) Guthrie, J . P. Can. J . Chem. 1979, 57, 236-239.

8154 J . Org. Chem., Vol. 63, No. 23, 1998
J ankowska et al.
from the gel with methanol. Fractions containing pure
products were collected and evaporated to dryness to yield
colorless, hygroscopic foams. For the 31P NMR data, see Table
1.
protocol involving phosphonylation or thiophosphonyla-
tion of standard nucleosidic precursors, followed by a
rapid deprotection with methylamine. The 31P NMR
studies on model compounds enabled us to delineate the
most crucial parameters affecting the sulfurization reac-
tion. The main advantages of the developed methods for
the preparation of nucleoside phosphoromonothioates and
nucleoside phosphorodithioates are the following: (i) The
phosphorothioate or phosphorodithioate function in nu-
cleotides 5 or 6 is generated in the last synthetic step,
and thus a possibility of its degradation is minimized.
(ii) The sulfurization is rapid and occurs under mild
reaction conditions. (iii) The purification procedure of the
products 5 and 6 by silica gel chromatography is simple,
fast, and compatible with a multigram scale syntheses.
(iv) The starting materials, nucleoside H-phosphonates
3 and nucleoside H-phosphonothioates 4, are stable, easy
to handle, and readily accessible from standard or
commercially available nucleosidic precursors.
2′-Deoxya d en osin -5′-yl H-p h osp h on a te, tr ieth yla m m o-
n iu m sa lt (3a ): yield 82%; RfThy ) 1.22 (A), 0.74 (B); 1H NMR
δH (D2O) 1.09 (9H, t, J ) 7.6 Hz), 2.21, 2.28 (2H, 2m), 3.15
(6H, q, J ) 7.6 Hz), 3.87 (2H, m), 4.05 (1H, m), 4.54 (1H, m),
1
6.26 (1H, t, J ) 6.6 Hz), 6.48 (1H, d, J HP ) 639.0 Hz), 7.97,
(1H, s), 8.18 (1H, s). HRMS (LSIMS, glycerol) [M-]: m/z
314.0669; calcd for [C10H13N5O5P]-, 314.0654.
2′-Deoxycytid in -5′-yl H-p h osp h on a te, tr ieth yla m m o-
n iu m sa lt (3b): yield 84%; RfThy ) 1.11 (A), 0.42 (B); 1H NMR
δH (D2O) 1.07 (9H, t, J ) 7.2 Hz), 2.14, 2.20 (2H, 2m), 2.99
(6H, q, J ) 7.2 Hz), 3.86 (2H, m), 3.98 (1H, m), 4.32 (1H, m),
5.88 (1H, d, J ) 7.6 Hz), 6.11 (1H, t, J ) 6.6 Hz), 6.54 (1H, d,
1J HP ) 638.4 Hz), 7.71 (1H, d, J ) 7.6 Hz). HRMS (LSIMS,
glycerol) [M-]: m/z 290.0535; calcd for [C9H13N3O6P]-, 290.0542.
2′-Deoxygu a n osin -5′-yl H -p h osp h on a t e, t r iet h yla m -
m on iu m sa lt (3c): yield 69%; RfThy ) 1.0 (A), 0.20 (B); 1H
NMR δH (D2O) 1.10 (9H, t, J ) 7.4 Hz), 2.35, 2.64 (2H, 2m),
3.03 (6H, q, J ) 7.4 Hz), 3.75 (2H, m), 4.05 (1H, m), 4.52 (1H,
1
m), 6.12 (1H, t, J ) 6.9 Hz), 6.65 (1H, d, J HP ) 639.6 Hz),
Some compounds prepared during the course of these
studies (i.e. 1d-f, 5d-f, 6d-f) were preliminarily screened
for a biological activity as thymidylate synthase inhibi-
tors.8 More studies on this subject are in progress.
7.86 (1H, s). HRMS (LSIMS, glycerol) [M-]: m/z 330.0613;
calcd for [C10H13N5O6P]-, 330.0603.
Th ym idin -5′-yl H-ph osph on ate tr ieth ylam m on iu m salt
(3d ): yield 74%; RfThy ) 1.0 (A), 1.0 (B); 1H NMR δH (D2O) 1.12
(9H, t, J ) 7.5 Hz), 1.75 (3H, m), 2.20, (2H, 2m), 3.03 (6H, q,
J ) 7.5 Hz), 3.91 (2H, m), 4.01 (1H, m), 4.21 (1H, m), 6.19
Exp er im en ta l Section
1
(1H, t, J ) 6.9 Hz), 6.61 (1H, d, J HP ) 637.5 Hz), 7.55 (1H,
m). HRMS (LSIMS, glycerol) [M-]: m/z 305.0545; calcd for
[C10H14N2O7P]-, 305.0539.
1H and 31P NMR spectra were recorded at 300 MHz
spectrometer. The 31P NMR experiments were carried out in
5 mm tubes using 0.1 mol/L concentration of phosphorus-
containing compound. Mass spectra were recorded with liquid
secondary ion mass technique (LSIMS) using Cs+ (12 keV) for
ionization. TLC analyses (Merck silica gel 60 F254 precoated
plates) were carried out in saturated chambers using the
following solvent systems: (A) i-PrOH/H2O/25% NH4OH (7:2:
1, v/v/v); (B) CH2Cl2/MeOH/Et3N (85:10:5, v/v/v). The RfThy
values reported are relative to thymidin-5′-yl H-phosphonate
3d and marked as RfThy. The amount of water in solvents was
measured with Karl Fisher coulometric titration. Methylene
dichloride was dried over P2O5, distilled, and kept over 4 Å
molecular sieves until the amount of water was less than 10
ppm. Pyridine was stored over 4 Å molecular sieves until the
amount of water was below 20 ppm. Triethylamine was
distilled and stored over CaH2. Trimethylsilyl chloride was
distilled before use. Elemental sulfur (pa grade) was used
without additional purification. For column chromatography
silica gel 60 (Merck) was used. Protected nucleoside 5′-H-
phosphonates15 1a -f and nucleoside 9-fluorenemethyl 5′-H-
phosphonothioates10 2a -f were prepared according to pub-
lished procedures.
2′-Deoxyu r id in -5′-yl H-p h osp h on a te, tr ieth yla m m o-
n iu m sa lt (3e): yield 88%; RfThy ) 0.98 (A), 0.83 (B);1H NMR
δH (D2O) 1.06 (9H, t, J ) 7.4 Hz), 2.18 (2H, 2m), 2.98 (6H, q,
J ) 7.4 Hz), 3.85 (2H, m), 3.95 (1H, m), 4.34 (1H, m), 5.70
1
(1H, d, J ) 8.1 Hz), 6.08 (1H, t, J ) 6.9 Hz), 6.26 (1H, d, J HP
) 638.4 Hz), 7.68 (1H, d, J ) 8.0 Hz). HRMS (LSIMS, glycerol)
[M-]: m/z 291.0375; calcd for [C9H12N2O7P]-, 291.0382.
5-F lu or o-2′-d eoxyu r id in -5′-yl H-p h osp h on a te, tr ieth y-
la m m on iu m sa lt (3f): yield 89%; RfThy ) 0.94 (A), 0.78 (B);
1H NMR δH (D2O) 1.11 (9H, t, J ) 7.6 Hz), 2.16 (2H, 2m), 3.07
(6H, q, J ) 7.5 Hz), 3.83 (2H, m), 3.94 (1H, m), 4.32 (1H, m),
1
6.08 (1H, t, J ) 7.2 Hz), 6.55 (1H, d, J HP ) 639.0 Hz), 7.83
3
(1H, d, J HF ) 6.3 Hz). HRMS (LSIMS, glycerol) [M-]: m/z
309.0285; calcd for [C9H11FN2O7P]-, 309.0288.
Gen er a l P r oced u r e for th e Syn th esis of Nu cleosid e 5′-
H-P h osp h on oth ioa tes 4a -f. A nucleoside 9-fluorenemethyl
H-phosphonothioate (2a -f, 1 mmol) in pyridine (5 mL) was
reacted with triethylamine (10 mL) for 20 min to remove the
9-fluorenemethyl group (TLC analysis). The solvent was
removed under vacuum and the residue treated with aqueous
methylamine (50%, 2 mL) during 1 h. After concentration to
a viscous oil, the mixture was dissolved in water (30 mL) and
extracted with CH2Cl2 (2 × 50 mL). The aqueous phase was
concentrated, dissolved in a minimum volume of CH2Cl2-
MeOH (4:1, v/v), and applied on a silica gel column. The
unprotected nucleoside H-phosphonothioates 4 were eluted
from the column using CH2Cl2-MeOH-Et3N (75:20:5, v/v/v).
Fractions containing pure compounds 4 were combined, evapo-
rated, and, after being dissolved in a minimum amount of
methanol, freeze-dried from benzene. White, amorphous,
hygroscopic powders were obtained. For the 31P NMR data,
see Table 1.
Gen er a l P r oced u r e for th e Syn th esis of Nu cleosid e
H-P h osp h on a tes 3a -f. A suitably protected nucleoside (3′-
O-benzoylthymidine, 3′-O-benzoyl-2′-deoxyuridine, 3′-O-ben-
zoyl-5-fluoro-2′-deoxyuridine, 3′-O-benzoyl-N2-isobutyryl-2′-
deoxyguanosine, 3′-O,N4-dibenzoyl-2′-deoxycytidine, or 3′-O,N6-
dibenzoyl-2′-deoxyadenosine, 1 mmol) was rendered anhydrous
by repeated evaporation of added pyridine (2 × 10 mL),
dissolved in the same solvent (10 mL) and treated with
diphenyl H-phosphonate (3 molar equiv, 15 min). The reaction
was quenched by the addition of water-triethylamine (1:1, v/v,
4 mL) and left for 15 min. The mixture was concentrated and
the residue treated with 50% aqueous CH3NH2 (10 mL) for 60
min. After evaporation of the solvents under vacuum, the oily
residue was partitioned between water (10 mL) and CH2Cl2
(10 mL). The aqueous layer was extracted with CH2Cl2 (2 ×
10 mL) and concentrated, and the residue, after being dis-
solving in a minimum volume of MeOH, was applied on a silica
gel column, equilibrated with CH2Cl2-MeOH (95:5, v/v). The
column was washed with CH2Cl2 containing Et3N (95:5, v/v),
and the unprotected nucleoside H-phosphonates 3 were eluted
2′-Deoxya d en osin -5′-yl H-p h osp h on oth ioa te, tr ieth y-
la m m on iu m sa lt (4a ): yield 82%; RfThy ) 1.17 (A), 1.36 (B);
1H NMR δH (D2O) 1.09 (9H, t, J ) 7.5 Hz), 2.42, 2.67 (2H,
2m), 3.01 (6H, q, J ) 7.5 Hz), 3.93 (2H, m), 4.10 (1H, m), 4.55
1
(1H, m), 6.27 (1H, t, J ) 6.9 Hz), 7.60, 7.62 (1H, 2d, J HP
)
1
593.2 Hz, J HP ) 594.0 Hz), 7.97, (1H, s), 8.25 (1H, 2s)
(multiplicity of some signals is due to the presence of P-
diastereoisomers); HRMS (LSIMS, glycerol), [M-] m/z 330.0428,
calcd for [C10H13N5O4PS]- 330.0426.
2′-Deoxycytid in -5′-yl H-p h osp h on oth ioa te, tr ieth yla m -
1
m on iu m sa lt (4b): yield 81%; RfThy ) 1.17 (A), 1.36 (B); H

Nucleoside H-Phosphonates
J . Org. Chem., Vol. 63, No. 23, 1998 8155
NMR δH (D2O) 1.15 (9H, t, J ) 7.5 Hz) 2.20, 2.29 (2H, 2m)
3.06 (6H, q, J ) 7.5 Hz), 4.02 (2H, m), 4.10 (1H, m), 4.44 (1H,
m), 5.96 (1H, d, J ) 7.6 Hz), 6.21 (1H, t, J ) 6.9 Hz), 7.76,
7.78 (1H, 2d, 1J PH ) 593.1 Hz), 7.83, 7.85 (1H, 2d, J ) 7.6 Hz)
(multiplicity of some signals is due to the presence of P-
diastereoisomers); HRMS (LSIMS, glycerol), [M-] m/z 306.0294,
calcd for [C9H13N3O5PS]- 306.0313.
2′-Deoxygu a n osin -5′-yl p h osp h or oth ioa te, a m m on iu m
sa lt (5c): yield 54%; RfThy ) 0.42 (A), 0.11 (B); 1H NMR δH
(D2O) 2.31, 2.53 (2H, 2m), 3.79 (2H, m), 4.02 (1H, m), 4.49 (1H,
m), 6.00 (1H, t, J ) 6.6 Hz), 7.93 (1H, s). HRMS (LSIMS,
glycerol) [M-]: m/z 362.0331; calcd for [C10H13N5O6PS]-,
362.0324.
Th ym idin -5′-yl ph osph or oth ioate, am m on iu m salt (5d):
1
2′-Deoxygu a n osin -5′-yl H-p h osp h on oth ioa te, tr ieth y-
la m m on iu m sa lt (4c): yield 88%; RfThy ) 0.92 (A), 0.44 (B);
1H NMR δH (D2O) 1.17 (9H, t, J ) 7.5 Hz), 2.42, 2.71 (2H,
2m), 3.09 (6H, q, J ) 7.5 Hz), 4.01 (2H, m), 4.14 (1H, m), 4.61
yield 72%; RfThy ) 0.64 (A), 0.70 (B); H NMR δH (D2O) 1.74
(3H, m), 2.17 (2H, 2m), 3.82 (2H, m), 3.97 (1H, m), 4.41 (1H,
m), 6.15 (1H, t, J ) 6.3 Hz), 7.66 (1H, m). HRMS (LSIMS,
glycerol) [M-]: m/z 337.0244; calcd for [C10H14N2O7PS]-,
337.0259.
1
(1H, m), 6.19 (1H, t, J ) 6.9 Hz), 7.69 (1H, d, J HP ) 594.9
Hz), 7.97, 7.99 (1H, 2s) (multiplicity of some signals is due to
the presence of P-diastereoisomers); HRMS (LSIMS, glycerol),
[M-] m/z 346.0354, calcd for [C10H13N5O5PS]- 346.0375.
Th ym id in -5′-yl H-p h osp h on oth ioa te, tr ieth yla m m o-
n iu m sa lt (4d ): yield 81%; RfThy ) 1.14 (A), 1.63 (B); 1H NMR
δH (D2O) 1.16 (9H, t, J ) 7.4 Hz), 1.82 (3H, m), 2.26 (2H, 2m),
3.17 (6H, q, J ) 7.4 Hz), 4.03 (2H, m), 4.09 (1H, m), 4.47 (1H,
m), 6.23 (1H, t, J ) 6.9 Hz), 7.62 (1H, m), 7.77, 7.79 (1H, 2d,
1J HP ) 592.5 Hz), (multiplicity of some signals is due to the
presence of P-diastereomers); HRMS (LSIMS, glycerol), [M-]
m/z 321.0311, calcd for [C10H14N2O6PS]- 321.0310.
2′-Deoxyu r idin -5′-yl ph osph or oth ioate, am m on iu m salt
(5e): yield %; RfThy ) 0.61 (A), 0.53 (B); 1H NMR δH (D2O) 2.14
(2H, 2m), 3.76 (2H, m), 3.94 (1H, m), 4.35 (1H, m), 5.70 (1H,
d, J ) 8.1 Hz), 6.08 (1H, t, J ) 6.9 Hz), 7.87 (1H, d, J ) 8.1
Hz). HRMS (LSIMS, glycerol) [M-]: m/z 323.0114; calcd for
[C9H12N2O7PS]-, 323.0103.
5-F lu or o-2′-d eoxyu r id in -5′-yl p h osp h or oth ioa te, a m -
m on iu m sa lt (5f): yield 94%; RfThy ) 0.58 (A), 0.50 (B); 1H
NMR δH (D2O) 2.23 (2H, 2m), 4.01 (2H, m), 4.11 (1H, m),
3
4.81 (1H, m), 6.21 (1H, m), 8.06 (1H, d, J HF ) 6.3 Hz).
HRMS (LSIMS, glycerol) [M-]: m/z 341.0001; calcd for
[C9H11FN2O7PS]-, 341.0009.
2′-Deoxyu r id in -5′-yl H-p h osp h on oth ioa te, tr ieth yla m -
1
m on iu m sa lt (4e): yield 92%; RfThy ) 0.97 (A), 1.50 (B); H
Gen er a l P r oced u r e for th e Syn th esis of Nu cleosid e
P h osp h or od ith ioa tes 6a -f. A nucleoside H-phosphonothio-
ate (4a -f, 1 mmol) and sulfur (3 molar equiv) were rendered
anhydrous by evaporation of added excess of pyridine (2 × 10
mL) and suspended in pyridine (10 mL) containing triethy-
lamine (20 molar equiv). When the mixture became homoge-
neous (ca. 10 min), trimethylsilyl chloride (5 molar equiv) was
added. After 15 min 0.1 M NaOH (7 molar equiv) was added,
and the solvent and excess reagents were evaporated under
vacuum. The resulting oily residue was triturated with
methanol (10 mL) and centrifuged off the precipitated salts,
and the methanolic solution containing 6 was evaporated.
Crude nucleoside phosphorodithioates 6 were dissolved in a
minimum volume of MeOH-concentrated NH4OH (9:1, v/v)
and applied on a silica gel column, preequilibrated with
2-propanol-H2O-concentrated NH4OH (90:5:5, v/v/v). The
column was washed out with i-PrOH-H2O-NH4OH concen-
trated (85:10:5, v/v/v), and the unprotected nucleoside phos-
phorodithioates 6 were eluted with MeOH-concentrated
NH4OH (9:1, v/v). The fractions containing pure compounds
6 were collected, evaporated, and freeze-dried from 1 M TEAB
buffer (pH 8.5). White, hygroscopic, amorphous powders were
obtained. For the 31P NMR data, see Table 1.
NMR δH (D2O) 1.11 (9H, t, J ) 7.5 Hz), 2.31 (2H, 2m), 3.04
(6H, q, J ) 7.5 Hz), 3.91 (2H, m), 4.05 (1H, m), 4.42 (1H, m),
5.74, 5.77 (1H, 2d, J ) 8.1 Hz), 6.16 (1H, m), 7.71, 7.72 (1H,
2d, 1J HP ) 593.4 and 594.3 Hz) 7.79, 7.82 (1H, 2d, J ) 8.1 Hz)
(multiplicity of some signals is due to the presence of P-
diastereoisomers); HRMS (LSIMS, glycerol), [M-] m/z 307.0129,
calcd for [C9H12N2O6PS]- 307.0154.
5-F lu or o-2′-d eoxyu r id in -5′-yl H-p h osp h on oth ioa te, tr i-
eth yla m m on iu m sa lt (4f): yield 83%; RfThy ) 0.92 (A), 1.42
(B); 1H NMR δH (D2O) 1.12 (9H, t, J ) 7.5 Hz), 2.24 (2H, 2m),
3.03 (6H, q, J ) 7.5 Hz), 3.98 (2H, m), 4.04 (1H, m), 4.41 (1H,
1
m), 6.14 (1H, m), 7.72, 7.74 (1H, 2d, J HP ) 594.0 and 594.6
Hz), 7.92, 7.93 (1H, 2d, 3J HF ) 6.6 and 6.3 Hz) (multiplicity of
some signals are due to the presence of P-diastereomers);
HRMS (LSIMS, glycerol), [M-] m/z 325.0063, calcd for
[C9H11FN2O6PS]- 325.0059.
Gen er a l P r oced u r e for th e Syn th esis of Nu cleosid e
P h osp h or oth ioa tes 5a -f. Nucleoside 5′-H-phosphonate 3
(1 mmol, prepared as above) and sulfur (3 molar equiv) were
rendered anhydrous by repeated evaporation of added pyridine
(2 × 10 mL) and suspended in the same solvent (10 mL)
containing triethylamine (20 molar equiv). To this, under
stirring, was added trimethylsilyl chloride (5 molar equiv), and
after 10 min the reaction mixture was cooled to ca. 0 °C and
quenched with 25% aqueous ammonia (1 mL). The reaction
mixture was concentrated to a small volume, and the oily
residue was partitioned between CH2Cl2 (10 mL) and water
(10 mL). The aqueous phase was extracted with CH2Cl2 (2 ×
10 mL), evaporated to dryness, and, after being dissolving in
a minimum volume of methanol-25% aqueous ammonia (95:
5, v/v), applied on a silica gel column, equilibrated with
2-propanol-concentrated ammonia-water (90:5:5, v/v/v). The
column was washed with the same solvent, and the unpro-
tected nucleoside phosphorothioates 5 were eluted from the
gel with methanol-25% aqueous ammonia (95:5, v/v). The
fractions containing pure products were collected and evapo-
rated to dryness to yield colorless, hygroscopic foams. For the
31P NMR data, see Table 1.
2′-Deoxya d en osin -5′-yl p h osp h or oth ioa te, a m m on iu m
sa lt (5a ): yield 79%; RfThy ) 0.81 (A), 0.32 (B); 1H NMR
δH (D2O) 2.37, 2.61 (2H, 2m), 3.84 (2H, m), 4.09 (1H, m), 4.53
(1H, m), 6.28 (1H, t, J ) 7.2 Hz), 8.00 (1H, s), 8.39 (1H, s).
HRMS (LSIMS, glycerol) [M-]: m/z 346.0383; calcd for
[C10H13N5O5PS]-, 346.0375.
2′-Deoxycytid in -5′-yl p h osp h or oth ioa te, a m m on iu m
sa lt (5b): yield 86%; RfThy ) 0.70 (A), 0.24 (B); 1H NMR δH
(D2O) 2.18 (2H, 2m), 3.90 (2H, m), 4.05 (1H, m), 4.42 (1H, m),
5.99 (1H, d, J ) 7.5 Hz), 6.16 (1H, t, J ) 6.9 Hz), 8.01 (1H, d,
J ) 7.5 Hz). HRMS (LSIMS, glycerol) [M-]: m/z 322.0290;
calcd for [C9H13N3O6PS]-, 322.0263.
2′-Deoxya d en osin -5′-yl p h osp h or od ith ioa te, d itr ieth y-
la m m on iu m sa lt (6a ): yield 75%; RfThy ) 0.77 (A), 1.22 (B);
1H NMR δH (D2O) 0,87 (18H, t, J ) 7.2 Hz) 2.38 (12H, q, J )
7.2 Hz) 2.47, 2.70 (2H, 2m), 3.98 (2H, m), 4.19 (1H, m), 4.65
(1H, m), 6.28 (1H, t, J ) 7.2 Hz), 8.10 (1H, s), 8.61 (1H, s).
HRMS (LSIMS, glycerol), [M-]: m/ z 362.0129; calcd for
[C10H13N5O4PS2]-, 362.0147.
2′-Deoxycytid in -5′-yl p h osp h or oth ioa te, d itr ieth yla m -
1
m on iu m sa lt (6b): yield 81%; RfThy ) 0.48 (A), 0.47 (B); H
NMR δH (D2O) 0.88 (18H, t, J ) 7.5 Hz) 2.25, (2H, 2m) 2.39
(12H, q, J ) 7.5 Hz), 3.99 (2H, m), 4.10 (1H, m), 4.49 (1H, m),
6.01 (1H, d, J ) 7.7 Hz), 6.24 (1H, t, J ) 6.9 Hz), 8.12 (1H, d,
J ) 7.7 Hz). HRMS (LSIMS, glycerol), [M-]: m/ z 338.0013;
calcd for [C9H13N3O5PS2]-, 338.0034.
2′-Deoxygu a n osin -5′-yl p h osp h or od ith ioa te, d itr ieth y-
la m m on iu m sa lt (6c): yield 77%; RfThy ) 0.24 (A), 0.28 (B);
1H NMR δH (D2O) 0.88 (18H, t, J ) 7.5 Hz), 1.15, 2.67 (2H,
2m), 2.34 (12H, q, J ) 7.5 Hz), 3.97 (2H, m), 4.15 (1H, m),
4.62 (1H, m), 6.21 (1H, m), 8.10 (1H, s). HRMS (LSIMS,
glycerol), [M-]: m/ z 378.0109; calcd for [C10H13N5O5PS2]-,
378.0096.
Th ym id in -5′-yl p h osp h or od ith ioa te, d itr ieth yla m m o-
n iu m sa lt (6d ): yield 75%. RfThy ) 0.44 (A), 1.49 (B); 1H
NMR δH (D2O) 0.74 (18H, t, J ) 7.3 Hz), 1.67 (3H, m),
2.06 (2H, 2m), 2.25 (12H, q, J ) 7.3 Hz), 3.84 (2H, m), 3.90
(1H, m), 4.33 (1H, m), 6.16 (1H, t, J ) 6.3 Hz), 7.48 (1H, m).
HRMS (LSIMS, glycerol), [M-]: m/ z 353.0023; calcd for
[C10H14N2O6PS2]- 353.0031.

8156 J . Org. Chem., Vol. 63, No. 23, 1998
J ankowska et al.
2′-Deoxyu r id in -5′-yl p h osp h or od it h ioa t e, d it r iet h y-
la m m on iu m sa lt (6e): yield 89%; RfThy ) 0.35 (A), 1.40 (B);
1H NMR δH (D2O) 1.14 (18H, t, J ) 7.5 Hz), 2.26 (2H, 2m),
3.07 (12H, q, J ) 7.5 Hz), 3.98 (2H, m), 4.11 (1H, m), 4.52
(1H, m), 5.82 (1H, d, J ) 8.2 Hz), 6.21 (1H, t, J ) 6.6 Hz),
8.10 (1H, d, J ) 8.2 Hz); HRMS (LSIMS, glycerol), [M-] m/ z
338.9873, calcd for [C9H12N2O6PS2]- 338.9875.
Ack n ow led gm en t. We are indebted to Prof. Maciej
Wiewio´rowski and Prof. Per J . Garegg for their interest
and helpful discussions. Financial support from the
State Committee for Scientific Research, Republic of
Poland, the Swedish Natural Science Research Council,
and the Swedish Foundation for Strategic Research is
gratefully acknowledged.
5-F lu or o-2′-d eoxyu r id in -5′-yl p h osp h or od ith ioa te, d it-
r iet h yla m m on iu m sa lt (6f): yield 76%; RfThy ) 0.26 (A),
1.41 (B); 1H NMR δH (D2O) 0.82 (18H, t, J ) 7.2 Hz), 2.10
(2H, 2m), 2.33 (12H, q, J ) 7.2 Hz), 3.92 (2H, m), 4.32 (1H,
Su p p or tin g In for m a tion Ava ila ble: NMR spectra (24
pages). This material is contained in libraries on microfiche,
immediately follows this article in the microfilm version of the
journal, and can be ordered from the ACS; see any current
masthead page for ordering information.
3
m), 4.76 (1H, m), 6.15 (1H, m), 7.84 (1H, d, J HF ) 6.0 Hz);
HRMS (LSIMS, glycerol), [M-] m/ z 356.9800, calcd for
[C9H11FN2O6PS2]- 356.9780.
J O980491U